Summary: Heme-mediated redox reactions have been suggested to contribute to organ dysfunction and/or tissue damage that occurs in some pathological states characterized by the release of hemoglobin or myoglobin into extracellular environment or when using cell free Hb-based blood substitutes. We investigated the pathways of cellular injury and death in normal and glutathione (GSH)-depleted bovine aortic endothelial cells subjected to bolus hydrogen peroxide (H2O2) or in the presence of a steady state levels of H2O2 generated by an enzymatic system. Diaspirin cross-linked hemoglobin (DBBF) an a analogue of a commercially produced product (DCLHb) was converted into its highly toxic, ferryl (Fe4+) form when this protein was incubated with endothelial cells treated with bolus amount of H2O2. The formation of Fe4+-DBBF was associated with significant decrease in endothelial cell GSH compared with the addition of H2O2 or Hb alone. There was distinct pattern of cell death (i.e. apoptosis vs.necrosis) associated with use of this Hb. Cell death and injury was more evident in cells without their own antioxidant mechanisms, i.e. GSH (D'Agnillo et al., Biochim Biophys Acta 1495, 150-159, 2000; D'Agnillo F, and Alayash AI. Am J. Physiol, in press). Enzymatically-generated H2O2 oxidized Ferrous Fe2+-DBBF and set off a redox cycling reaction between Ferric (Fe3+) and Fe4+-DBBF. Cells subjected to DBBF and enzymatically-produced H2O2 induced sever cellular rounding, swelling, and detachment, and accumulated in the G2/M phase of cell cycle. This G2/M arrest proceeded the onset of apoptosis as revealed by increased phosphatidylserine (PS) externalization and DNA fragmentation measured by annexin V assay and sub G1/G0 events, respectively. Once again G2/M arrest and apoptosis was more pronounced in endothelial cells depleted of their intracellular GSH. These redox reactions of DBBF seen in our endothelial cell model systems may explain the greater toxicity observed with its commercial analogue (DCLHb). Baxter a leading developer of these products recently terminated its clinical development due to the unexpected high death rate in trauma patients infused with this product.